RU2027757C1 - Method of in vitro plant-regenerants preparing - Google Patents

Abstract

FIELD: plant biotechnology, tissue and cell culturing, microcloning. SUBSTANCE: plants - regenerants were prepared in vitro. For regeneration increase acetone is added (concentration is 10000-20000 mg/l) additionally to the nutritional media for callus initiation and regenerant forming. EFFECT: improved method of plant-regenerants preparing.

Description

 The invention relates to biotechnology, namely to obtain regenerates in vitro.

 The development of biotechnology methods, which are based on the cultivation of isolated organs, tissues and cells on artificial nutrient media, followed by the regeneration of whole plants, determines their increasing introduction into agricultural practice. They are able to significantly accelerate the breeding process and significantly expand the boundaries of human exposure to wildlife.

 In the development of in vitro methods, where regenerants are required for introduction into the selection process, they deal with the emergence of meristematic zones from callus and the formation of the latter from plants. This technique is necessary, first of all, for microcloning of plants, as well as for obtaining somaclonal variability and when working on selective media, including any selective factor (salt, herbicides, toxins of phytopathogenic fungi, etc.).

There are studies in the literature on the development of methods for the formation of cereal regenerants, in particular wheat from callus tissue of the embryo [1-2]. Using these methods, the embryos are isolated from immature kernels and planted on a nutrient medium to initiate callus. Cultivation planted embryos is carried out in the dark at t = 26 ^o C. Material browsing every 2-3 days. in order to remove the main seedling in case of its growth and thereby contribute to the formation of callus on the surface of the scutellum. Visually, callus formation is noted at 6-10 days, however, the growth of the main seedling inhibits this process and it has to be broken off at least 2 times, which greatly complicates the work.

 Callus growth on the scutellum can occur within 1-3 weeks. and more. During this time, visible dense foci are formed on calluses, differing from loose hydrated callus in consistency and color. Callus with dense areas is morphogenic, and when it or only one dense areas are transplanted onto the regeneration medium, green leaf-like structures are formed first, and after: 2-3 weeks. seedlings. It should be noted that with the onset of the morphogenetic process, the dense region, which is a cluster of embryonic cells, is easily divided into separate units (embryoids), each of which gives a seedling, also planted separately.

 From explant landing on nutrient media and to the release of regenerated plants, the work takes place in three stages: 1. - callus induction and meristematic zones; 2. - the formation of regenerants; 3. - growing plants and accelerating them before planting in the ground. At each of these stages of the medium, they differ in the replacement of some additives with others, in their quantity and ratio. This replacement allows you to direct the process either towards callus formation or organogenesis with the development of roots or regenerants.

In the process of developing an optimal environment for obtaining regenerants from callus of wheat, the universal medium Murashige and Skoog was selected, manufactured by the industry in finished form, which includes the following components, mg / l: macro salts: NH ₄ NO ₃ 1650; KNO ₃ 1900; MgSO ₄ ˙ 7H ₂ O 370; CaCl ₂ ˙ 2H ₂ O 440; KH ₂ PO ₄ 170; microsols: MnSO ₄ ˙H ₂ O 22.3; H ₃ BO ₃ 6.2; ZnSO ₄ ˙ 7 H ₂ O 8.6; CoCl ₂ ˙6 H ₂ O 0.025; CuSO ₄ ˙5 H ₂ O 0.025; NaMoO ₄ ˙2 H ₂ O 0.25; KI 0.83; vitamins: thiamine 0.1-10.0; mininozitol 80-100; pyridoxine 0.5-1.0; nicotinic acid 0.5-1.0.

Based on the stages of work on obtaining regenerated plants on the specified medium, the following additives for each stage are intended for the cultivation of cereals, in particular wheat, mg / l:
Wednesday I (for the induction of callus and the formation of meristematic zones).

 Wednesday Murashige and Skoog, sucrose 20000-30000, mg / l, agar-agar 6000-7000 mg / l. Dichlorophenoxyacetic acid (2,4-D) 1.5-2.5 mg / L.

 Wednesday II (for the formation of regenerants).

 Murashige and Skoog medium, sucrose 10000-15000 mg / l, agar-agar 6000-7000 mg / l, α-naphthylacetic acid (ANU) and kinetin 0.5-1.0 mg / l.

 Wednesday III (for growing plants and rooting them).

 Wednesday Murashige and Skoog, sucrose 10000-15000 mg / l, agar-agar 6000-7000 mg / l. No additives.

 When immature cereal embryos, in particular wheat, are planted, mass regeneration of plants takes place. Nevertheless, the number of regenerated plants varies greatly, primarily, depending on the genotype, but also on the composition of the culture media.

 The aim of the invention is to develop a method for producing plant regenerants in vitro, giving a greater number of plants, especially on selective media.

 This goal is achieved using the in vitro method of producing regenerants, including planting an explant on a nutrient medium for initiation of callus and laying of meristematic zones, growing seedlings when transplanting meristematic zones on a nutrient medium for processing them into regenerants, as well as growing and accelerating plants before transplanting into the ground , the hallmark of which is that acetone in an amount of 10000-20000 mg / l is additionally introduced into the media for the initiation of callus and the formation of regenerants.

 The use of acetone in nutrient media for the production of higher plant regenerants has not been described in the scientific, technical and patent literature, which suggests that the proposed method meets the criteria of the invention of "novelty."

As you know, acetone belongs to the group of ketone bodies, which includes acetone, acetoacetic acid and β-hydroxybutyric acid. In the body, acetone breaks down into CO ₂ and H ₂ O.

 The method of obtaining regenerates in vitro on media containing acetone, has been tested on various crops, including wheat, onions, garlic, cabbage and canola.

 PRI me R 1 (control). Immature wheat germ was cultured on a nutrient medium of the following composition, mg / l: macro and micro salts according to Murasiga and Skoog, sucrose 30000, agar-agar 7000; vitamins: thiamine 10, myoinositol 100, pyridoxine and nicotinic acid at 1.0, 2.4-D 2.5 (in the medium for the initiation of callus or meristematic zones) or ANU and kinetin at 1.0 (in the medium for regeneration). pH 5.6-5.8.

 On Wednesday I immature wheat germ is planted with its shield upward at the age of 10-14 days after pollination of 20 test tubes of each sample, 6 seeds in each tube - a total of 120 seeds of each sample. 1-2 days after planting, the planted main seedling begins to grow, which should be broken off. After another 3-7 days, it is necessary to view the tubes and remove the remaining growth of the main shoot. In 7-10 days after planting, the formation of callus and its subsequent growth are noted on the surface of the scute. Depending on the genotype, the percentage of callus formation is 76-85% of the number of planted embryos. Calli are on this medium for 21-28 days before the appearance of dense patches - meristematic foci, after which the dense patches are transferred to medium II, where 4-8 weeks are spent on them. seedlings are formed depending on the genotype in 25-30% of cases. The callus remaining after the transfer is planted on medium I for further callus growth and the formation of new meristems in it.

 It is possible to cultivate immature wheat germ on Murashige and Skoog medium with the following content, mg / l: sucrose 20,000, agar-agar 60,000, thiamine 1.0, myoinositol 80, pyridoxine and nicotinic acid 0.5, 2.4-D 1.5 (in the medium for the initiation of callus and meristematic zones) or ANU and kinetin at 0.6 (in the medium for regeneration). pH = 5.6-5.8.

 On these media, results similar to those given above were obtained, with the difference that with a decrease in the amount of 2,4-D from 2.5 to 1.5 mg / L, an acceleration in time of meristematic zone formation occurs during 2-3 days. These data make it possible to formulate the main (basic) medium I and II for further work, mg / l: Murasige and Skoog medium, agar-agar 6500, sucrose 30000, thiamine 10.0, myoinositol 100, pyridoxine and nicotinic acid 0.5 2,4-D (in medium I) 2.0 or ANU and kinetin (in medium II) 0.5 and kinetin 1.0.

 The resulting seedlings were transferred to medium III for growth and acceleration. There was no difference in growth and rooting on media composed to the maximum or minimum (sucrose 15,000 mg / L, agar-agar 7,000 mg / L or sucrose 10,000 mg / L, agar-agar 6,000 mg / L. Therefore, for growing and rooting, Murashige and Skoog medium containing sucrose 12000 mg / l and agar-agar 6000 mg / l (basic medium III) can be recommended as the best option.

Well-developed test tube plants were planted in the ground, where they produced seeds. It should be noted the violation in some ears of regenerated plants (P _o ) of the fertility of some flowers, which disappeared in plants of the first generation (P ₁ ).

 PRI me R 2 (experience). Immature wheat germ is cultured on a medium with acetone of the following composition, mg / l: basic medium I or II plus acetone 5000, 10000, 20,000 and 50,000.

 Immature embryos of the same wheat samples are cultivated as described above (Example 1) with the shield facing upwards at the age of 10-14 days after pollination with 20 test tubes of each variant, 6 embryos in each test tube - a total of 120 embryos of each sample. Viewing plantings after 2-3 days found that the growth of the main seedling is inhibited: the seedlings do not go to growth at all or grow weakly and in the extreme case they need to be broken off once, which greatly facilitates the work. In this effect - the first significant advantage of environments with acetone.

 The further development of callus is intensive and within 12-15 days meristematic zones are laid in it, which, after transferring them to the medium for regeneration, form regenerants in many. The number of meristematic zones on a medium with acetone is 87-95% relative to the number of planted embryos. 53–65% of regenerants were obtained on this medium. It should be emphasized the high activity and continuity of the process of laying the meristematic zones and the associated increase in the number of regenerants in the medium with acetone compared to the control. This effect is the second significant advantage of acetone media for in vitro production of wheat regenerants from callus tissue.

 The cultivation of immature embryos on a base medium with acetone of 5000, 10000, 20,000 and 50,000 mg / l was tested. The best options for the yield of callus, meristematic zones and regenerants should be considered options with an acetone content of 10,000 and 20,000 mg / l, while there was no fundamental difference between these options. With an acetone content of 5000 mg / L in the medium, a weak effect was observed. The cultivation of embryos on a medium with an acetone content of 50,000 mg / L led to the death of explants, therefore this option is not taken into account.

Regenerants obtained on medium II grew and rooted in base medium III and planted in the ground where they gave seeds. It should be emphasized that the fertility of P _o plants in the soil in the variant of 10,000 and 20,000 mg / l of acetone was, on the whole, almost equal to that in the control, but in individual plants there were up to 50% sterile flowers in ears. As in the control, P ₁ plants completely restored fertility. In addition, the plants R _o and R ₁ noted a high bushiness (10-30, stems), while the bulk of the shoots were productive. In control, bushiness was common for spring wheat - 2-5 shoots. In the high bushiness of the experimental spring wheat plants, which makes it possible to obtain in P _{1 a} larger grain yield from one bush, this is the third advantage of acetone media for obtaining wheat regenerants from callus tissue in vitro.

 Example 2 makes it possible to recommend an environment with acetone for wheat of the following composition: base medium plus acetone 15000 mg / L.

 It should be noted that these studies have been tested experimentally for 3 years, with unequivocal results. Due to the extremely interesting data, the method was also tested on an in vitro onion culture during the formation of regenerants and similar results were obtained.

PRI me R 3 (control). The tissues of the bottom of the onion bulbs are cultivated with the aim of microcloning them for the rapid propagation of new varieties and valuable breeding samples, as well as for the vegetative propagation of F ₁ -F ₂ hybrids (when it is required to preserve them without splitting) or sterile interspecific hybrids.

 Since the bottom of the onion contains meristematic cells, microcloning does not require the formation of callus and the laying of meristematic zones in it, therefore, medium I is abolished. The explant is planted on medium II (for regeneration), represented by a base medium containing instead of kinetin 2.5-3.5 mg / l benzylaminopurine (BAP).

For planting, the bulb is exempted from dry scales, sterilized, washed with sterile water, the first row of juicy scales is removed and cut so that the bottom with the remains of juicy scales (no more than 0.5 cm long) can be cut into 5-6 sections (0.5 x 0.5 cm). Each plot is planted on a nutrient medium. It should be noted that explants taken from the peripheral areas of the bottom give more regenerants than those taken from the central areas. Cultivation takes place in the light at 10-15 tys.pks light, day length of 16 hours and t = 20-25 ° ^C. After 2-3 weeks the first regenerants appear as thin green needles, which, as their appearance Free lemmas and starting from tissues and transplanted either on medium II for the growth of new regenerants, or on medium III for growing and rooting. It was shown that when explants were cultured on medium II containing 2.5 or 3.5 mg / l BAP, there was no fundamental difference in terms of the yield of regenerants. Therefore, the optimal environment is recommended - basic + BAP 3.0 mg / l. It is shown that from 1 explant on this medium can be obtained within 2.5-3.5 months. 50-70 plants, and work on microcloning can be carried out at any time of the year.

 PRI me R 4 (experience). The above method is used to cultivate the tissue of the onion bottom on the base medium + BAP 3.0 mg / l + acetone 10000-20000 mg / l. There was no fundamental difference in the development of regenerants between the variants of 10,000 and 20,000 mg / l of acetone in the medium. Therefore, the medium with acetone for onion microcloning should be considered optimal, mg / l: base medium + BAP 3.0 + acetone 20000. On this medium, the number of regenerants increased by 3.5 times, which amounted, depending on the genotype, to 2.5 -3.5 months 150-300 regenerants from one explant.

 Similar data were obtained by microcloning of garlic.

PRI me R 5. It is known that the regeneration of various varieties and varieties of cabbage is difficult, which is a big obstacle to the work in vitro and genetic engineering methods with this object. Therefore, an experiment was performed to obtain regenerants on a medium with acetone. For this, the cotyledons of white cabbage seedlings of the varieties Amager and June 10, as well as broccoli, after sterilization were planted on agar medium II, represented by the base medium and containing 2.5-3.5 mg / l BAP instead of kinitin. Cultivation took place in the light at 10-15 tys.lks light, day length of 16 hours. And t = 20-25 ° ^C. It is shown that these media in any case not landing explants were regenerated. Therefore, the cotyledons were landed on medium II with a BAP content of 3.0 mg / L and acetone supplemented with 10000-30000 mg / L. On the medium with acetone 20,000 mg / L, the maximum number of regenerants was observed: in Amager variety, the number of explants with regenerants was 10% (in control 0%), in June variety 10 - 80% (in control 0%), in broccoli - 11% (in control 0%). When planting explants on a medium with acetone 10,000 mg / l, there were practically no regenerants. When planted on media containing 30,000 mg / l, the regenerants were somewhat disfigured (with twisted leaves). Thus, the environment with acetone should be considered optimal for obtaining cabbage regenerants from cotyledons: base medium + BAP 3.0 mg / l + acetone 20,000 mg / l.

 When planting a hypocotyl of cabbage seedlings on an optimal medium with acetone, broccoli obtained 44% of explants with regenerants, in the control - 13%.

 Similar results were obtained when in vitro planting rapeseed tissue.

 Thus, on the basis of the above material, we can conclude that the introduction of acetone in a nutrient medium has an advantage in the technique of producing regenerants in vitro.

Claims (1)

 METHOD FOR PRODUCING PLANTS - REGENERATORS IN VITRO, including planting an explant on a nutrient medium for initiation of callus and laying of meristematic zones, obtaining seedlings when transplanting meristematic zones on a nutrient medium for the formation of regenerants, growing and rooting plants on a nutrient medium before planting them in the ground that in a nutrient medium for the initiation of callus and the formation of regenerants, acetone is additionally introduced in an amount of 10000-20000 mg / l.